Heterocyclic cations, developed by our collaborative research groups, bind specifically to extended AT sequences in the DNA minor groove. These compounds have shown clinical biological activity with low human toxicity against several parasitic microorganisms, and an orally available prodrug has progressed through Phase II clinical trials against several parasitic organisms. The compound is scheduled to begin Phase III trials against human trypanosomiasis in October 2004. Biophysical and in vivo biological studies clearly suggest that the target of action of the compounds is DNA and, in particular, the kinetoplast DNA (kDNA) of kinetoplastid microorganisms, such as trypanosomes and leishmania. The heterocyclic cations have shown in vivo ability to block transcription enhancer binding to AT rich sequences and to redistribute the topoisomerase II enzyme, which is critical for replication of the unique kDNA system into AT rich regions. Our hypothesis is that both of these effects require synergistic action of closely bound drug molecules in the AT rich kDNA. The research in this proposal is designed to develop new DNA-targeted antimicrobial drugs by providing a better understanding of the molecular basis of the biological action of the dications in parasites. We will use both parallel and combinatorial chemistry approaches to develop systematic and rational sets of new compounds to address specific questions about the heterocycle-DNA complexes. A battery of traditional, as well as novel biophysical methods, will be used to evaluate the interactions of the compounds with different DNA sequences, such as those that they target in the microorganisms. We are specifically focused on the effects on DNA structure and protein inhibition of cooperative binding of the compounds at closely spaced AT rich binding sites. Both long and short range effects of the compounds on DNA structure and properties will be evaluated. Detailed structural studies will be conducted with selected compounds that we feel can provide fundamental insight into the molecular recognition mechanisms. We will test the ability of the compounds to inhibit transcription enhancers that selectively target AT rich sequences by using model peptide systems, as well as in vivo analysis of protein inhibition. All new compounds will be tested against several microorganisms, and the results will be correlated with their ability to bind DNA and inhibit protein-DNA interactions and functions.